Pediatr. Res. 14: 1212-1215 (1980)
cystic fibrosis
lung
macrophages
neutrophils
Pulmonary Nonspecific Defense Mechanisms in
Cystic Fibrosis
I. Phagocytic Capacity of Alveolar Macrophages
and Neutrophils
RAMON J. J. CASSINO, DANIEL 0 . SORDELLl, CARLOS N. MACRI, M A R C E L 0 KOHAN,
MARIO H. DILLON, A N D OMAR H. PIVETTA"'"
Centro Nacional de Genetica Medica, Combate de 10s Pozos 2193, and Hospital de Niios "Dr. Ricardo Gulierrez." S.
Bustamante 1399 Buenos Aires, Argenlina
Summary
Cystic fibrosis (CF) is the commonest cause of chronic obstructive lung disease within the first three decades of life. Because
patients suffer from repeated pulmonary infections, we were interested in studying the nonspecific antibacterial mechanisms of the
lungs of C F children. Fifteen C F children, 13 nontuberculous
pulmonary-infected (I) children and 4 noninfected (N-I) children,
who were diagnosed a s having a foreign body in the air passages,
were submitted to a bronchopulmonary washing. The absolute
number, viability, and differential count of the cells were determined. and the phagocytic assay using the Lehrer's technique was
carried out on the harvested cells.
The materials obtained from C F and I patients were mucopurulent, whereas the ones from the N-I patients were not mucopurulent. The number of cells retrieved from C F and I patients was
higher than that from the N-I patients. This was attributed to an
increase in the absolute number of polymorphonuclear leukocytes.
l'he phagocytic capacity of polymorphonuclear leukocytes was not
different for the three groups studied, whereas that of alveolar
macrophages was higher for the C F and the I patients than for the
N-I ones. Inasmuch as there was no significant difference between
the C F and the I patients, we conclude that the nonspecific
phagocytic behavior of alveolar macrophages and pulmonary polymorphonuclear leukocytes of C F patients might be similar to the
one coming from other bacterial infectious states of the lung.
Speculation
Although cystic fibrosis children suffer from chronic progressive
pulmonary infections, they show the normal population risk for
other infections outside the lungs. Inasmuch as the immune system
appears to be intact in these patients, a failure in one of the
nonspecific antibacterial defense mechanisms of the lung may be
considered.
The major cause of morbidity and mortality in cystic fibrosis
( C F ) is unquestionably the severe, chronic progressive pulmonary
infections due to bacterial pathogens. particularly Pseudomonas
aeruginosa and Staph~~lococcus
aureus (18). An important feature
of these infections is their striking localization in pulmonary tissue.
with only very rare spread via the bloodstream to other sites of
the body. Although this fact would tend to suggest that there is
not a primary genetic defect in immunologic defense mechanisms
in C F (4. 1 I), the overwhelming role of infection in the disease
has led to an exhaustive search for acquired or local pulmonaryimmunologic defect in this inherited disease.
The nonrespiratory activity of purifying inhaled air and keeping
lung tissues free of infection has been collectively termed "lung
host-defense mechanisms" (5); that includes mechanical barriers,
mucociliary clearance, immunoglobulins, and inflammatory response involving polymorphonuclear leukocytes (PMN). In addition, the alveolar macrophages (AM) play a principal role during
phagocytosis in the airways and during their scavenging activity
on the alveolar surfaces (6).
Several studies (7, 17) have suggested the compartmentalization
of cell-mediated immunity, i.e., both respiratory tract and systemic
cell-mediated immunity exist and are relatively independent of
each other. This concept would suggest an hereditary or acquired
immunologic defect localized solely in C F lung.
Because of our interest in the lung defense mechanisms, the
present study was undertaken to investigate the specific question
of whether there are any defects in pulmonary AM and PMN
nonspecific phagocytic function in CF.
SUBJECTS A N D METHODS
SUBJECTS
Fifteen C F patients, aged I to 17 years (9 females and 6 males)
were studied with a diagnostic or therapeutic proposal; C F was
diagnosed by clinical signs and by elevated sweat electrolytes;
clinical grading according to Shwachman and Kulczycki score
ranged from 65 to 90. All the children were on normal diets and
recGving appropriate therapy. Thirteen control subjects, aged 1 to
13 years (6 females and 7 males), were investigated for variable
reasons and were subsequently found to have nontuberculous
infectious pulmonary involvement and no positive sweat test. Four
children who had no lung infection were also investigated; these
subjects were diagnosed as patients with foreign body in airways;
none received antibiotics for at least 48 hr before bronchopulmonary lavage, and none was found to have a positive sweat test or
C F familiar history.
These studies were carried out with the proper consent from
parents of the children involved, according to the principles of the
Declaration of Helsinki.
C E L L RETRIEVAL
Under light general anesthesia achieved by inhalation of halothane (19). a rigid bronchoscope was introduced into the trachea
and the main stem bronchi. A radiopaque catheter was passed
through the bronchoscope as deeply as to the tip wedged into a
segmental or subsegmental airway within a zone which previously
had not shown any pathologic involvement to X-rays.
Aliquots of sterile 0.15 M NaCl solution were alternatively
instilled and withdrawn by gentle aspiration with a syringe. The
total volume of washing solution to be instilled was determined
according to the age and the lung size of the patient, calculated
from radiologic parameters (range, 50 to 200 ml). The complete
lavage procedure required approximately 5 min, and there were
no complications during or after performing it.
group and the N-I patients (Table 1). The total number of free
cells harvested by bronchial lavage (Table I) was higher in the I
patients and was attributed principally to a near 200-fold increase
in the absolute number of PMN, as is shown from the proportion
PROCEDURE
All samples of lung washing suspension were shaked vigorously
during 2 min; a 5-ml aliquot was treated with 1 ml of a 20%
solution of N-acetylcystein (20) and incubated 10 min at 37OC
with constant shaking. The total number of cells, the viability as
assessed by exclusion of trypan blue dye, and the differential cell
count on smears obtained by sedimentation and Giemsa staining
were determined.
The remainder of the material was sedimented 2 min at 1 x g
and decanted; the decanted solution was centrifuged 5 min at 200
x g. The viability of these cells was not different from those of the
N-acetylcystein-treated cells. From the pellet obtained, 4 x 10"
cells were scattered in Leighton tubes in I ml of tissue culture
medium (21) added to 10% of fresh human serum obtained from
a pool of normal subjects.
After 3 hr incubation, the adhered cells were rinsed twice with
warm tissue culture medium, and varying concentrations of Cand i d ~albicans were added to give a ce1l:yeast ratio of 1:4, 1:6, 13,
1: 12. and 1:16. In this way. a modified phagocytic assay of Lehrer
(9) was carried out by incubating the cell-yeast mixture for 30
min. The results of phagocytic capacity were expressed for the two
principal phagocytic cellular types (AM and PMN) as the number
of Candida yeasts clearly engulfed per 100 cells.
When a pair of means for samples distributed normally had to
be compared, a Student's t test for independent samples was
applied with significant levels of P < 0.01 and P < 0.05. If the
samples were not distributed normally and there was no homoscedasticity, the Mann-Whitney U test was applied (14).
RESULTS
The recovery of washing solution was more effective in CF
patients than in the noninfected (N-I) ones; the scattering of the
data from nontuberculous pulmonary-infected (I) individuals
made impossible the detection of any difference between this
I
Table 1. Percentage of wash fluid recovered and total cell content
and cell viability in wash fluid obtained by bronchopulmonary
lavage in CF children and control subjects
.Patients
N-l (n
=
4)
C F (n = 15)
Range
j?
Viability (%)
I
96.2
Range
i
Recovery of
29.80 20.0-38.0
wash fluid (%)
Cell no. ( x 10")
3.32 1.0-9.3
88-95
45.25' 27.5-58.8
l (n
=
13)
j?
Range
40.20
22.5-75.0
49.63' 14.463.98'
138.0
90.4
80-100 89.1
1.5144.0
70-97
Significantly different from the N-I patient group ( P < 0.01).
' Significantly different from the N-1 patient group ( P < 0.05).
Table 2. Differential cell count made on Giemsa-stained smears
obtained by sedimentation of samples of lung wash suspensions
Patients
N-I (n = 4)
i
AM
Foamy
cells
Giant
cells
PM N
Lymphocytes
Eosinophils
I
84
4
C F (n = 15)
Range
j?
76-89
1-10
16'
2
Range
347
1-6
l (n = 13)
i
Range
20'
1-56
1 0 - 4
Significantly different from N-l patient group ( P < 0.01)
PMN
C
cell
Fig. I. Relationship between the number of intracellular Candida per 100 lung cells ( I C C ) and the number of yeast particles per cell. cultured for
30 min (Cper cell). A , heavy mean values of the ICC engulted by both AM and PMN together: B and C, mean values of the ICC engulfed by PMN and
AM, respectively. In all cases, i S.D.
*
3 ET AL.
of these cells and AM (Table 2). The viability of the morphologically conserved cells (Table 1) was the same in the three groups;
however, it must be noted that all the infected (CF and I) patients
showed a high number of partially destroyed cells which were not
included in the viability counting.
The phagocytic capacity of the total content of free cells in
washing fluid was plotted against the inverse of the cel1:yeast ratio,
and the results are shown in Figure 1. Each point represents the
heavy average of the number of cell-associated yeasts found in
100 phagocytic cells, namely AM and PMN. Recognition of the
cell-associated Candida was easy, as shown in Figure 2.
Estimated values of phagocytic capacity overlapped in both
infected (CF and I) patients, whereas all the N-I patients showed
a lesser phagocytic capacity than the infected ones. This difference
was not detected when the PMN were considered separately (Fig.
1B); in other words, the phagocytic capacity of PMN was not
different for the three groups. However, the difference appeared
again when the phagocytic capacity of the AM was studied in
isolation (Fig. 1C). It can be noted that the differences were
detected between infected (CF and I) and noninfected patients
and only attributed to the AM.
DISCUSSION
During the last years, the involvement of AM in the antibacterial defense of the lungs (12) has been recognized as one of the
most important mechanisms. Because C F patients suffer from
persistent lung infection, the study of the lung defense mechanisms
in these children has become more important each day, and the
investigation in this field related to AM becomes a necessity. In
this context, different approaches have been worked out, although
the interest of these authors arose primarily in the study of the
influence of possible phagocytic inhibitory factors in C F serum
(2, 16) or in the decrease of serum factors which normally promote
the engulfment of particles by normal nonhuman AM (1). Other
studies have been developed to study the mechanisms of intracellular degradation of mucus by AM in C F patients; these results
have not been confirmed yet (3, 10).
The present work was directed towards the analysis of the
phagocytic capacity of AM and PMN harvested by bronchopulmonary washing of C F children.
Both the C F and the I patients had mucus hypersecretion,
which was not only seen macroscopically but detected through the
recovery of the wash solutions; this hypersecretion was less in NI patients than in the other two groups. Because the mucopurulent
samples obtained were difficult to process, it was necessary to
liquefy the suspensions with a mucolytic agent, whereas nontreated cells partially homogenized by shaking were preferred for
functional studies.
As it has been described previously, both C F and I patients
showed marked hypercellularity in lung-washing suspensions.
This was due to PMN (13, 15). In view of the free cell content of
airways, C F does not differ from other nontuberculous infectious
states.
Because separation and purification of respiratory cells constitute a considerable technical problem, we decided to apply the
method described by Lehrer (9) for evaluating simultaneously the
nonspecific phagocytic capacity of AM and PMN.
When the total cell content was analyzed for phagocytic capacity, no significant differences between C F and I patients were
found, and in both cases, the cells were more effective than those
from N-I patients. This clear difference may be well understood
in terms of macrophage activation, which was produced in vivo in
the infected lung environment. When AM and PMN were investigated separately, however, it was seen that this increase in
phagocytic capacity was associated with AM only. The mean
values of phagocytic capacity tend to be lower for C F than I
patients in almost all the assays performed; however, the differences were not significant, probably due to the dispersion of the
data. The results, therefore, indicate that the behavior of CF-AM
might not be substantially different from those of the AM found
in the airways of patients with nontuberculous infection.
The AM is the only tissue macrophage readily available for
study, and therefore, it has been the main source of the relatively
limited information on hand on macrophage function in human
tissues (8). The present study deals only with the phagocytic cells
which can adhere in 3 hr and remain adherent during the assay
procedure. Therefore, the results presented here should be carefully interpreted because the population of cells investigated may
not be either the predominant one or representative of the in vivo
whole organ.
Future studies regarding the evaluation of other CF-AM-specific mechanisms would provide further information related to the
pathogenesis of the C F lung bacterial involvement.
REFERENCES A N D NOTES
Fig. 2. Phagocytic cells of the respiratory tract of CF children. A , two
PMN which have engulfed yeasts of C. albicans (4 and 3 yeasts in left and
right PMN's, respectively); B, alveolar macrophage with one intracellular
yeast; C, binucleated giant cell with 3 engulfed yeasts (sedimented cells
stained with Giemsa, x 1250).
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22. The authors thank Dr. Tetsuji Matayoshi for his photographical advice and
Susana Paz for her secretarial assistance.
23. Requests for reprints should be addressed to: Omar H. Pivetta. MD., Centro
Nacional de Genetica Medica. Combate de los Pozos 2193. 1245 Buenos Aires.
Argentina.
24. Received for publication January 25, 1980.
25. Accepted for publication May 14. 1980.
Prinred in U. S. A.